The patterns and extent of vagal preganglionic divergence and convergence within the gastrointestinal tract of the rat were characterized with the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Three weeks after tracer was iontophoretically injected into two to four sites within the dorsal motor nucleus of the vagus, wholemounts of perfused gut organs (stomach, duodenum, cecum) were prepared, counterstained with Cuprolinic blue, and processed for PHA-L using the avidin biotin complex with diaminobenzidine. Controls included animals injected with PHA-L after intracranial deafferentations. Well-positioned injections labeled an extremely dense and intricate network of varicose efferent axons throughout the gastric myenteric plexus (including that of the fundus). Individual fibers collateralized extensively, forming a variety of pericellular arborizations and terminal complexes made up of both en passant and end swellings. Single axons frequently innervated subsets of neurons within ganglia. Most enteric neurons were contacted by varicosities of more than one vagal fiber. The patterns of vagal preganglionic fibers in the duodenal and cecal myenteric plexuses resembled the organization in the stomach in many aspects, but the projections in each organ had distinctive characteristics, and label was less dense in the intestines than in the stomach. Vagal preganglionic fibers directly innervated submucosal ganglia, although sparsely. Brainstem injections of PHA-L retrogradely labeled a few myenteric neurons in the corpus, fundus, and duodenum: These "gastrobulbar" and "duodenobulbar" neurons received reciprocal vagal preganglionic innervation. Finally, the PHA-L that spread to the nucleus of the solitary tract occasionally produced transganglionic labeling of afferent intramuscular arrays (gastric fundus). The results of this paper provide strong evidence that the traditional "command neuron" or "mother cell" hypotheses of vagal-enteric organization should be abandoned for an integrative neural network model.

Three-dimensional reconstruction protocols in confocal microscopy are typically considered in terms of rendering separate stacks of optical sections. Single stacks, however, include volumes that are often too small to permit descriptions of entire neurons, complete axonal arbors, or complex neural networks. Furthermore, traditional tissue preparation protocols generally yield specimens too limited to permit reconstructions of complex neural systems. For 3-D analyses of extensive networks such as the autonomic nervous system projections within the viscera, it is critical to incorporate appropriate tissue techniques, including suitable tracer protocols, into the reconstruction strategy. This report summarizes complementary technologies, including whole mount procedures, tracer techniques for identifying single fibers in situ, and methods of examining stacks of optical images, which make it practical to describe the complete terminal field of an individual axon in the gastrointestinal tract. Such methods establish that vagal motor axons travel long distances within their target organs, collateralize frequently, and ramify extensively. Vagal afferents have extensive, complex, and, in some cases, polytopic arbors within target tissues.